PROJECT D ? Electron Microscopy of the Class III Phosphatidylinositol 3-Kinase Complex in Autophagy James Hurley PROJECT SUMMARY/ABSTRACT Autophagy is a central pathway for cellular homeostasis and robustness to stress. The synthesis of phosphatidylinositol 3-phosphate (PI(3)P) by the autophagy-specific class III phosphatidylinositol 3-kinase complex (PI3KC3) is fundamental and central to the initiation of autophagy. Normal autophagic function protects agains neurodegenerative diseases. Autophagy is also tumor suppressive, since the protein encoded by the BECN1 tumor suppressor gene is part of the core autophagy machinery. Declines in autophagic function are associated with aging and neurodegeneration. The development of allosteric PI3KC3 activators would appear to have excellent potential as therapeutic stimulators of autophagy. The principal form of PI3KC3 involved in triggering autophagy consists of a 1:1:1:1 complex of the subunits VPS34, VPS15, BECN1, and ATG14. The structures of many individual ordered fragments of PI3KC3 have been determined, but there are We have now obtained a 3D reconstruction of PI3KC3 for the first time using negative stain EM. The complex is dynamic, however, with careful attention to the selection of appropriate class average and the use of maximum likelihood-based 3D reconstruction, we have been able to generate a 28 map. We have used maltose-binding protein tags to identify all of the subunits, and all of the known ordered domains have been assigned to regions of the density. We will carry forward the initial negative stain reconstruction into cryoelectron microscopy to extend the resolution as high as possible, with potential to reach atomistic resolution. CryoEM will then be used to understand in detail the structural changes that occur when PI3KC3 is activated by starvation, by phosphorylation by kinases such as ULK1, and by binding to regulatory proteins such as NRBF2. Insights into the allosteric circuitry of PI3KC3 activation will be invaluable conceptually for development of autophagy activators. High resolution cryoEM structures may ultimately have a direct practical application in identifying the binding sites and mechanisms of action of lead compounds. The work outlined here will, if successful, have a transformative impact on the basic science of autophagy initiation and that therapeutic targeting of autophagy to fight neurodegenerative diseases, cancer, and aging. no structural data on the larger complex.